An updated systematic review and meta-analysis on Mycobacterium tuberculosis antibiotic resistance in Iran (2013-2020)

This updated systematic review and meta-analysis follows two aims: 1) to assess Mycobacterium tuberculosis (M. tuberculosis) antibiotic resistance in Iran from 2013 to 2020 and, 2) to assess the trend of resistance from 1999 to 2020. Several national and international databases were systematically searched through MeSH extracted keywords to identify 41 published studies addressing drug-resistant M. tuberculosis in Iran. Meta-analysis was done based on the PRISMA protocols using Comprehensive Meta-Analysis software. The average prevalence of resistance to first- and second-line anti-TB drugs, multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) in new and previously treated tuberculosis (TB) cases in Iran during 2013–2020 were as follows: isoniazid 6.9%, rifampin 7.9%, ethambutol 5.7%, pyrazinamide 20.4%, para-aminosalicylic acid 4.6%, capreomycin 1.7%, cycloserine 1.8%, ethionamide 11.3%, ofloxacin 1.5%, kanamycin 3.8%, amikacin 2.2%, MDR-TB 6.3% and XDR-TB 0.9%. Based on the presented data, M. tuberculosis resistance to first- and second-line anti-TB drugs, as well as MDR-TB, was low during 2013–2020 in Iran. Furthermore, there was a declining trend in TB drug resistance from 1999 to 2020. Hence, to maintain the current decreasing trend and to control and eliminate TB infection in Iran, continuous monitoring of resistance patterns is recommended.


Introduction
On 24 March 1882, Dr Robert Koch discovered a rod-shaped, aerobic bacterium called Mycobacterium tuberculosis (M. tuberculosis). It is the causative agent for tuberculosis (TB) which is one of the ten most common causes of death worldwide. The latent form of tubercle bacilli has been detected in 1.7 billion people around the world (1-3). Between 5 and 10% of these people will eventually develop the active form of the disease (pulmonary or extrapulmonary) in their lifetime (4,5). TB is an air-borne and communicable disease and humans are the only natural reservoir of the infection (6). According to the latest report of the World Health Organization (WHO) in 2018, TB remained a global health problem with 10 million infected people (57% adult men, 32% adult women, and 11% children), 1.2 million deaths in HIV-negative and 251,000 deaths in HIV-positive patients (1). In 2014, WHO announced "the End TB Strategy" which aimed to reduce TB-associated deaths (by 90%) and incidence rate (by 80%) and to end the global TB epidemic between 2016 and 2035 (1). To achieve these purposes, developing novel diagnostic tests for rapid detection, use of effective drugs or new treatments, and appropriate vaccination are needed (1). Since 1921, the bacilli Calmette-Guérin (BCG) live attenuated vaccine has been the only approved vaccine for TB prevention in newborns and children. It has variable protection against adult pulmonary TB (0-80%) while it is unable to prevent the reactivation of latent TB (7,8). Treatment with first-line drugs i. e., isoniazid, rifampin, ethambutol, and pyrazinamide is recommended for a six-month period in drugsusceptible TB as the mortality rate is high without treatment (1). The success rate for first-line drugs is >85%, however, the emergence of drug-resistant M. tuberculosis, particularly multidrug-resistant TB (MDR-TB defined as resistance to isoniazid and rifampin) has led to a decrease in treatment success to 56%, an increase in treatment duration and costs, and administration of more toxic second-line drugs (1). TB incidence in 2018 in Iran, a country with a population of 82 million located in the Middle East, showed a decreasing trend (11, 000; 14 cases per 100,000 population), however, it has shared geographical borders with high incidence countries such as Pakistan (6% in 2018) (1). Hence, continuous surveillance on TB epidemiology especially drug resistance status is of high necessity for disease control. M. tuberculosis antibiotic resistance pattern has previously been studied between March 1999 and May 2013 in Iran (9). Nevertheless, as the prevalence of TB antibiotic resistance is changing over time, we have performed the current systematic review and meta-analysis from 2013 to 2020 to update the evidence in Iran.

Search strategies and data sources
The current systematic review and meta-analysis was conducted based on the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analysis) guidelines (10

Data selection criteria and quality assessment
The titles, abstracts, and full texts of collected articles were further evaluated in detail to select eligible studies based on our inclusion and exclusion criteria. In addition, the Joanna Briggs Institute (JBI) critical appraisal checklist was selected to assess the quality of each included article. Eligible studies showed high quality (>5 scores), medium quality (4-5 scores), or low quality (<4 scores) (11). Original articles assessing the prevalence of M. tuberculosis antibiotic resistance with the following features were included in the study: (1) published in English or Persian, 2) limited to Iran, 3) clinical strains of M. tuberculosis isolated from new or previously treated cases, 4) studies evaluating monoresistance (which is defined as resistance to only 1 first-or second-line anti-TB drugs), MDR and XDR (extensively drug-resistant) status and 5) full-text availability. The following studies were excluded from analysis: 1) studies reporting drug resistance patterns in nontuberculous mycobacteria (NTM), 2) studies that only focused on drug resistance mechanisms, 3) studies that solely evaluated any drug resistance status (which is defined as resistance to any drug regardless of monoresistance), 4) non-original studies, 5) duplicate publications including same studies published in both English and Persian languages and two or more relevant studies conducted by the first or corresponding author in the same year and also several multicenter studies with identical information such as same authors' name, same enrollment time and previously determined antibiotic resistance profiles, 6) studies available only in abstract form and dissertations, 7) studies without sufficient data such as study date or ambiguous information, 8) studies performed merely to evaluate the sensitivity and specificity of different antimicrobial susceptibility testing methods using previous banks of bacterial isolates or clinical isolates of M. tuberculosis with known drug resistance profiles, 9) studies on M. tuberculosis other than antibiotic resistance such as epidemiological patterns or treatment of the disease, and 10) multicenter studies without sufficient and separate data for each province.

Data extraction
Extracted information from eligible studies included in the meta-analysis is shown in Table 1. Important data were as follows: first author's surname, publication date, region of study, study enrollment date, number of tested isolates, methods used for assessing bacterial antibiotic susceptibility, and prevalence of M. tuberculosis resistance to first-and second-line drugs.

Data analysis
Meta-analyses were conducted using the Comprehensive Meta-Analysis (CMA) (Biostat, Englewood, NJ) software package, and antimicrobial resistance trends were determined by the Graphpad Prism software package. Important analyzed parameters included the rates of M. tuberculosis antibiotic resistance and heterogeneity and publication bias among studies. The pooled estimates of the resistance prevalence for each drug were reported as a percentage and 95% confidence intervals (CIs) using a random-effects model (heterogeneity ≥25%;) or fixed-effects model (heterogeneity <25%;). The possibility of heterogeneity was evaluated by I² statistic and the Chi-square test with the Cochrane Q statistic. The existence of publication bias was assessed by Funnel plots.

Results
As shown in Figure 1, a total of 1,354 articles in Persian and English languages were collected during early literature search. At the end of the screening, 41 eligible articles were included in the meta-analysis    (Table 1). The most common laboratory methods that were used for assessing M. tuberculosis antibiotic susceptibility were agar proportion and molecular techniques such as line probe assay (LPA), real-time polymerase chain reaction (PCR), multiplex allele-specific polymerase chain reaction (MAS-PCR), and multiplex PCR. The total prevalence of isoniazid-resistant M. tuberculosis among new and previously treated cases in Iran was 6.9% (95% CI: 5.5-8.6) between 2013 and 2020, which was estimated using a random-effects model due to existence of significant heterogeneity (I 2 = 92.1%; Q = 1101.5; P = 0.00). As shown in Table  2, the highest prevalence of isoniazid-resistant M. tuberculosis strains in different provinces of Iran was found in Fars (16.7%) and the lowest rates were seen in Semnan (0%), Yazd (0%), and Zanjan (0%). Additionally, Figure 2  Semnan (0%), and Zanjan (0%) showed the highest and lowest rates of resistance to rifampin, respectively. Among new and previously treated cases between 2013 and 2020, the mean resistance to ethambutol in Iran was 5.7% (95% CI: [4][5][6][7][8]. The rate of resistance was highest in Kermanshah (14.2%) and lowest in Golestan (0%). The overall prevalence of ethambutol-resistant M. tuberculosis strains in Iran was calculated using the random-effects model (I 2 = 86%; Q = 208.3; P = 0.00). As presented in Figure 2, ethambutol-resistant strains also showed a decreasing pattern in Iran from 1999 to 2020 (9.7% to 5.7%). Frequency of M. tuberculosis resistance to pyrazinamide calculated using the fixed-effects model was 20.4% (95% CI: 15.7-26.2; I 2 = 13.2%; Q = 11.5; P = 0.31). The highest resistance rate was found in Hamadan (40%) and the lowest rate was seen in Razavi Khorasan (0%). Second-line anti-TB drugs (groups A-D) used for MDR treatment include 1) group A: fluoroquinolones (ofloxacin, levofloxacin, moxifloxacin, and ciprofloxacin), 2) group B: injectable drugs (aminoglycosides (kanamycin, amikacin, and streptomycin) and cyclic peptides (capreomycin)), 3) group C: ethionamide/ prothionamide, cycloserine/terizidone, linezolid and clofazimine, and 4) group D: D2 (bedaquiline and delamanid) and D3 ( para-aminosalicylic acid, imipenem-cilastatin, meropenem, amoxicillin/ clavulanate and thioacetazone). The prevalence of M. tuberculosis resistance to these drugs which are mainly

Discussion
Despite the strategies initiated by the National Tuberculosis Control Program (NTP) in 1996, TB continues to be a major health concern in Iran. One of the main reasons is the emergence of drug-resistant M. tuberculosis strains (53). The WHO implemented a general project back in 1994 in which data on anti-TB drug resistance are collected from numerous countries; the results indicate that the prevalence of drug-resistant M. tuberculosis strains is still a major public health concern around the world (1). The current metaanalysis is the newest report on the M. tuberculosis antibiotic resistance in Iran. We believe it provides such overviews of the available data to estimate the total antibiotic resistance. Also, it helps to increase our knowledge on drug-resistant TB trends during the years to prevent further rise in drug resistance and treatment failures. Among the first-line anti-TB drugs recommended by NTP to treat new sputumpositive TB cases in Iran, isoniazid and rifampin are the most effective agents (16). Isoniazid has also been recommended as a preventive therapy in latent TB infections (54). Therefore, isoniazid-and rifampinresistant cases can increase the risk of treatment failure due to development of MDR-TB. Our analysis showed that 6.9% and 7.9% of both new and previously treated cases between 2013 and 2020 in Iran were resistant to isoniazid and rifampin, respectively. Based on the WHO report in 2018, the global average of isoniazid resistance varies between 7.2% among new TB cases and 11.6% in previously treated TB cases (1). Additionally, findings of the present study on the prevalence of rifampinresistant strains of M. tuberculosis (7.9%) is comparable with international data from the Western Pacific Region (24%), European Region (10%), South-East Asian Region (6%), African Region (3%) and Region of America (1%) (55). In 2018, WHO announced that the incidence of MDR-TB and rifampicin-resistant TB in new and previously treated TB cases were 3.4% and 18% in the world, respectively (1). The rate of MDR-TB in Iran was low (6.3%). Patients with rifampicin-resistant TB and MDR-TB must be treated with second-line drugs   (1). Among first-line anti-TB agents, the highest and lowest resistance rates between 2013 and 2020 in Iran were seen for pyrazinamide (20.4%) and ethambutol (5.7%). It has been suggested that chromosomal mutations are associated with development of drug resistance in clinical M. tuberculosis strains including rifampicin resistance-associated mutations in rpoB gene, mutations in katG or inhA genes in isoniazid-resistant strains, embB or ubiA genes mutations in ethambutolresistant isolates, and mutations in pncA, rpsA, panD and clpC1 genes among pyrazinamide-resistant isolates (56). Similar resistance mechanisms were seen in M. tuberculosis isolated in Iran (data not shown).
Furthermore, the prevalence of resistant strains to isoniazid, rifampin, ethambutol, and streptomycin as well as MDR-TB in Iran showed a downward trend from 1999 to 2020 (Figure 2). This could be attributed to differences in the methods used for assessing drug susceptibility/antibiotic resistance due to possible heterogeneity among studies (fixed-or random-effects models) or the number of included studies among two reviews. Also, decreased incidence of TB cases in Iran (21 per 100,000 population in 2011 compared with 14 cases in 2018) and fewer Afghan refugees due to sanctions, are other contributory factors. The limitations that need to be acknowledged in this study include: 1) small number of studies reporting resistance to secondline drugs and XDR-TB, 2) lack of studies reporting M. tuberculosis drug resistance in some provinces, 3) lack of individual separation of drug-resistant TB in many studies according to new or previously treated patients, age, ethnicity, nationality (Iranian versus Afghans), and 4) existence of heterogeneity and publication bias among included studies.

Conclusion
The current updated systematic review and metaanalysis summarized the total prevalence of drugresistant M. tuberculosis strains in new and previously treated TB cases (2013-2020) and drug-resistant TB trend (1999-2020) in Iran. Based on our results, the mean resistance to first-and second-line anti-TB drugs was low in Iran during 2013-2020 as were the cases of MDR-TB and XDR-T. There was also a decreasing trend in resistance of M. tuberculosis from 1999 to 2020. Hence, to continue the current downward trend and control and eliminate TB infections in Iran, continuous monitoring of resistance patterns through optimized and rapid diagnostic tests such as molecular techniques in all provinces of Iran is recommended.

Conflicts of Interest
The author declares that there are no conflicts of interest.